A Consistent PCR-RFLP Assay Based on ITS-2 Ribosomal DNA for Differentiation of Fasciola Species.

Objective(s): Fascioliasis is a zoonotic parasitic disease caused by liver fluke species of Fasciola hepatica and Fasciola gigantica. Differentiation of these two species, based on their morphological characteristics, is difficult. The current study aimed to use PCR-RFLP assay to distinguish between F. hepatica and F. gigantica, based on profiles of RFLP, produced by effect of endonucleases on ITS2 of the ribosomal DNA genes from these two species. Materials and Methods: Adult Fasciola spp. were isolated from bile duct of naturally infected animals. The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene in the isolates. By running the sequences of the samples in NEBcutter, suitable restriction enzymes (MspI and KpnI) were selected. Eight F. gigantica and eighteen F. hepatica samples were evaluated. Results: While RFLP pattern with MspI produced a profile by which it was difficult to differentiate these two species, KpnI along with MspI, produced a consistent pattern of a 231, 212 and 93 bp fragments in F. hepatica. This pattern was not seen in F. gigantica. Conclusion: Findings of this study demonstrated that RFLP with KpnI and MspI produce a suitable pattern which simply differentiates F. hepatica from F. gigantica.


Introduction
Fascioliasis is a zoonotic parasitic disease caused by the liver fluke species of the genus Fasciola. F. hepatica and F. gigantica are two species which infect human and animals. F. hepatica has a worldwide distribution and both species are pres ent in the tropical and s ubtropical regions of Africa and Asia (1).
Differentiation of thes e two s pecies, based on thei r morphological characteristics, is difficult. The differences in the intermediate hosts, control strategies, transmission patterns and also epidemiological characteristics which overlap in some area i ndicate that the proper differenti ation of F. hepatica and F. gigantica infections i n human or animals is crucial (2).
Due to the limitations of morphological methods, several molecular approaches, using different molecular targets, have been developed for the differentiation of F. hepatica and F. gigantica (3).
Several DNA-bas ed approaches have been used for differentiation of Fasciola species (4-7). Among them, sequencing of the first and the second internal transcribed s pacers (ITS-1 and ITS-2) of ribosomal DNA and mitochondrial DNA (mtDNA) provided reliable genetic markers for species -level identification of Fasciola species (3). ITS-2 sequence is located between the 5.8S and 28S codi ng regions of rDNA that are highly conserved and hav e few inter-specific nucleotides useful for genetic characterization and identification in both F. hepatica and F. gigantica (8)(9).
Although sequencing of ribosomal or mitochondrial DNA which shows the differences between the nucleotides of the desired gene, offers reliable methods for differentiation of Fasciola species, RFLP-based approaches provide a relatively simple, cost-effective and appropriate method for differential diagnosis of F. hepatica and F. gigantica. In line with this, different restriction enzymes and target genes hav e been used (2). The current study was performed to examine the utility of a PCR-RFLP assay for differentiation of F. hepatica and F. gigantica, based on RFLP profiles, produced by the effect of endonucleas es on ITS2 of the ribosomal DNA genes from these two species.

Parasite
Adult Fasciola spp. were isolated from bile duct of naturally infected sheep, goat and cattle at the slaughterhouses from various regions of Kohgiluyeh and Boyer-Ahmad province in Iran, where human cases of fasciolosis has been recently reported (10). Flukes were washed extensively in PBS (37°C) and subsequently fixed in 70% ethanol and maintai ned at 4°C for s everal weeks until used.

DNA extraction and polymerase chain reaction (PCR)
For genomic DNA extraction, a portion of the apical and lateral zone of adult flukes was removed and crushed. DNA from the crushed materials was extracted with phenol-chloroform method. Briefly, 500 μl of lysis buffer and 8 μl of protei nase K was added to the sample and incubated overnight at 37°C. Afterward, 100 μl of phenol-chloroform was added and centrifuged at 1000 g for 10 mi n at 25°C. Top aqueous phas e was removed and absolute alcohol was used to precipitate the DNA. Extracted DNA was diluted in doubl e distilled water and pres erved at 4°C until used.

Restriction fragment length poly morphism (RFLP)
After sequencing, using NEBcutter V2.0 software (12), the cutti ng sites of commercially available restriction enzymes on ITS2 sequences of F. hepatica and F. gigantica were assessed (Figure 1). MspI and KpnI were selected as the enzymes which might produce the most informative profile. For RFLP, a total volume of 20 μl, including 10 μl of ITS2 PCR product was added with 1 μl of ei ther MspI or KpnI, 4 μl of 10x Tango buffer (Fermentas, Lithuania) and 4 μl of DD-H2O. The tubes were incubated at 37°C for 12 hr, accordi ng to the manufacturer instruction to ensure full cutting of fragments. For analyzing the digestion products, 15 μl of each product in addition to 2 μl of loading buffer were run in 2% gel electrophoresis.

Results
The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene i n the isolates. All amplified products of both F. hepatica and F. gigantica were diges ted with the MspI restriction endonuclease. Eight F. gigantica and eighteen F. hepatica samples were evaluated. RFLP pattern with MspI produced a 212 and 324 bp fragments for F. hepatica and 218 and 322 bp for F. gigantica (Figure 2). Bas ed on these profiles, it was quite difficult to differentiate these two species. Using KpnI along wi th MspI (double digestion), a consistent pattern was found where KpnI cut ITS2 fragment of F. hepatica and produced a 231, 212 and 93 bp fragments (Fi gure 3). This pattern was not seen in F. gigantica ( Figure 3  for genetic characterization and identification of parasites especially helminthes (13,14). In the present study, a rapid and simple method of RFLP assay, based on the partial rDNA of ITS2 gene, was utilized for the accurate differentiation and identification of Fasciola species.
Restriction enzymes are powerful and simpl e approaches toward the characterization of parasite species based on differences in their genomes. These methods have been used for differentiation among Fasciola species based on the profiles generated by the effects of endonucleas es on ITS genes of these parasites (4,5). In a study in China, effect of Hsp92II or RcaI on ITS2 region, Fasciola spp. were differentiated from one another by their unique restriction patterns (8). Isolates of Chinese Fasciola produced a mixture of patterns in two Fasciola species. Huang et al (2004) us ed the res triction endonucleases, Hsp92II and RcaI, on ITS2 region for molecular differentiation of Fasciola s pp. and showed that Hsp92II produces different profiles in different isolates from different hosts. They reported that Hsp92II is better than RcaI for differentiating between thes e two species (8).
Fasciola species have been traditionally classified based on their morphological features, such as width and body length. Because of the size variations of thes e two species, the discrepancy of morphological features, and the presence of intermedi ate forms, it is difficult to distinguish the two species, solely based on these characteristics. RFLP is a powerful approach for discriminating thes e two species. RFLP pattern have been used for characterization of Fasciola species and also other organisms in Iran (15)(16)(17)(18). Karimi (2008) showed that in 18S DNA region, BfrI restriction enzyme produce similar profile for both F. hepatica and F. gigantica whereas DraI generates different patterns for two species of Fasciola (15). Rokni et al (2010) used TasI res triction enzyme for ITS1 region which properly differentiated F. hepatica from F. gigantica (2). In another study, Saki et al (2011) showed that AvaII and DraII restriction enzymes in 28S DNA appropriately differentiate these two speci es (16). However, Ghavami et al (2009) showed that the pattern of restriction di gestion in ITS2 sequence of Fasciola samples which was seen with BamHI and PagI restriction enzymes at the nucleotide positions of 230, 340 and 341 bp are specific to F. hepatica species and has no effect on F. gigantica (17).

Conclusion
In the current study, considering the s equences of ITS2 of F. hepatica and F. gigantica, two endonucleases, MspI and KpnI, were us ed to differentiate Fasciola spp. For MspI, the RFLP profiles of two species were very similar while for KpnI the profile was quite different and this enzyme was able to cut the ITS2 of two species of Fasciola at different sites which may be utilized for the differenti ation of two species. The evaluation of the Fasciola spp. in the areas where two species of the fluke coexist is important and this simple RFLP can be used for discerning these two species.